Part:BBa_K5136230

I0500-B0034-mt3-B0034-mt2a-B0015

Biology

MT3 and MT2A

The metallothioneins (MTs) are a class of low molecular weight and cysteine-rich metal binding proteins, and each one of them can bind to 6-9 heavy metal ions. The MTs are expressed as intracellular proteins and are primarily responsible for metal regulation in cells of living organisms. General MTs can widely non-covalently bind divalent heavy metal ions, such as Zn²⁺, Ni²⁺, Pb²⁺, Hg²⁺, Cd²⁺, as well as As³⁺, but their effectiveness in treating Cr₂O₇^2- is not satisfactory. MT2A and MT3 are metallothioneins(MTs) found in Homo sapiens. MT2A not only has efficient adsorption capacity for ordinary metal ions, but also exhibits efficient processing capacity for Cr₂O₇^2-. And MT3 has a better adsorption effect on ordinary metal ions. (1).

Usage and Design

To absorb Cd²⁺ in waste water, we introduced MntA membrane protein in E. coli, which translocates external Cd²⁺ into the cell. To increase the strain's tolerance to Cd²⁺, we constructed this composite part(BBa_K5136230) to produce intracellular MT2A and MT3 in engineered bacteria, mitigating the cell damage of Cd²⁺.

Characterization

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136230) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli BL21(DE3). The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (2106 bp) can be observed at the position around 2000bp (Figure 1).

Figure 1 Colony PCR of BBa_K5136230_pSB1C3 in E. coli BL21(DE3)

Reference

1. A. A. Uçkun, M. Uçkun, S. Akkurt, Efficiency of Escherichia Coli Jm109 and Genetical Engineering Strains (E. Coli MT2, E. Coli MT3) in Cadmium Removal from Aqueous Solutions. Environ. Technol. Innovation 24, 12 (2021).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 1239
  Illegal BamHI site found at 1472
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961